Two 5S rDNA arrays in Neotropical fish species: is it a general rule for fishes?

In this paper we describe Southern blot hybridization results probed with 5S rRNA genes for several Neotropical fish species representing different taxonomic groups. All the studied species showed a general trend with the 5S rDNA tandem repeats organized in two distinct size-classes. At the same time, data on 5S rDNA organization in fish genome were summarized. Previous information on the organization and evolution of 5S rRNA gene arrays in the genome of this vertebrate group are in agreement with the Southern results here presented. Sequences obtained for several fish species have revealed the occurrence of two distinct 5S rDNA classes characterized by distinct non-transcribed spacer sequences, which are clustered in different chromosomes in some species. Moreover, the 5S rDNA loci are generally distributed in an interstitial position in the chromosomes and they are usually not syntenic to the 45S rDNA. The presence of two classes of 5S rDNA in several non-related fish species suggests that this could be a common condition for the 5S rRNA gene organization in the fish genome.     

Molecular organization of 5S rDNA in fishes of the genus Brycon.

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.     

Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers.

To address understanding the organization of the 5S rRNA multigene family in the fish genome, the nucleotide sequence and organization array of 5S rDNA were investigated in the genus Leporinus, a representative freshwater fish group of South American fauna. PCR, subgenomic library screening, genomic blotting, fluorescence in situ hybridization, and DNA sequencing were employed in this study. Two arrays of 5S rDNA were identified for all species investigated, one consisting of monomeric repeat units of around 200 bp and another one with monomers of 900 bp. These 5S rDNA arrays were characterized by distinct NTS sequences (designated NTS-I and NTS-II for the 200- and 900-bp monomers, respectively); however, their coding sequences were nearly identical. The 5S rRNA genes were clustered in two chromosome loci, a major one corresponding to the NTS-I sites and a minor one corresponding to the NTS-II sites. The NTS-I sequence was variable among Leporinus spp., whereas the NTS-II was conserved among them and even in the related genus Schizodon. The distinct 5S rDNA arrays might characterize two 5S rRNA gene subfamilies that have been evolving independently in the genome.     

The 5S ribosomal RNA gene in Pythium species: two different genomic locations.

The 5S ribosomal RNA (rRNA) genes in eukaryotes may occur either interspersed with the other rRNA genes in the ribosomal DNA (rDNA) repeat, or in separate tandem arrays, or dispersed throughout the genome. In Pythium species and in several related Oomycetes, polymerase chain reaction (PCR) amplification of the nontranscribed spacer (NTS) region with one primer specific for the 5S gene revealed, with several exceptions, that the 5S rRNA gene was present in the rDNA repeat of those species with filamentous sporangia and was absent from the rDNA repeat of those with globose or unknown sporangia. When present, the gene was located approximately 1 kb downstream of the large-subunit rRNA gene and on the strand opposite that on which the other rRNA genes were located. This was confirmed in P. torulosum by sequencing of the gene and its flanking regions. The 5S rRNA genes of P. ultimum were found in tandem arrays outside the rDNA repeat. Evidence of dispersed 5S rRNA sequences was also observed. For many of the species of Pythium having the 5S gene in the NTS, the region between the large-subunit rRNA gene and the 5S gene was found to have length heterogeneity. Oomycetes related to Pythium were also found to have the 5S gene in the NTS, although sometimes in the opposite orientation. This may mean that the presence of the gene in the NTS is ancestral for the Oomycetes and that the absence of the gene in the NTS in those Pythiums with globose sporangia is due to loss of the gene from the rDNA repeat in an ancestor of this group of species. These results favor the view that 5S rRNA gene linkage to the rRNA cistron existed prior to the unlinked arrangement seen in most plants and animals.     

Molecular organization of 5S rDNA in bitterlings (Cyprinidae)

Molecular organization and nucleotide sequences of the 5S rRNA gene and NTS were investigated in freshwater fish, bitterlings (Acheilognathinae), including 10 species/subspecies of four genera, Acheilognathus, Pseudoperilampus, Rhodeus, and Tanakia, to understand the evolutionary trait of 5S rDNA arrays. Southern hybridization analysis revealed a general trend with tandem repeats of 5S rDNA in all the examined bitterlings. Sequence analysis demonstrated a conserved 120 bp sequence of the 5S rRNA gene and a short NTS of 56–67 bp with two distinct portions, a conserved (5′-flanking portion; at positions −1 to −38) and a variable part (3′-flanking portion), in 6 of 10 species/subspecies examined. The conserved NTS region was most likely an external promoter so far observed in various vertebrates, whereas the variable NTS region could be divided into two types due to its nucleotide polymorphisms. Molecular phylogeny using the 5S rRNA gene and NTS sequences suggested the occurrence of 5S rDNA duplication before speciation and a concerted evolution for the gene and conserved NTS regions, but a birth-and-death process to maintain the variable NTS region. Thus, the 5S rDNA in the examined bitterlings might have evolved under a mixed process of evolution.     

Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome

Background

The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat.

Results

Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70–80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors.

Conclusion

A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread wheat has been established. These two regions differ in the organization of both 5S rDNA and the neighboring sequences comprised of transposable elements, implying different modes of evolution for these regions.

     

Restriction enzyme analysis of Bacillus subtilis ribosomal ribonucleic acid genes.

The organization of the ribosomal ribonucleic acid (rRNA) genes (rDNA) of Bacillus subtilis was examined by cleaving the genome with several restriction endonucleases. The rDNA sequences were assayed by hybridization with purified radioactive rRNA's. Our interpretation of the resulting electrophoretic patterns is strengthened by an analysis of a fragment of B. subtilis rDNA cloned in Escherichia coli. The results indicated that there are eight rRNA operons in B. subtilis. Each operon contains one copy of the sequences coding for 16S, 23S, and 5S rRNA. The sequences coding for 5S rRNA were shown to be more closely linked to the 23S rRNA genes than to the 16S rRNA genes.     

5S rDNA variation and its phylogenetic inference in the genus <Emphasis Type="Italic">Leporinus</Emphasis> (Characiformes: Anostomidae)

5S rDNA sequences have proven to be valuable as genetic markers to distinguish closely related species and also in the understanding of the dynamic of repetitive sequences in the genomes. In the aim to contribute to the knowledge of the evolutionary history of Leporinus (Anostomidae) and also to contribute to the understanding of the 5S rDNA sequences organization in the fish genome, analyses of 5S rDNA sequences were conducted in seven species of this genus. The 5S rRNA gene sequence was highly conserved among Leporinus species, whereas NTS exhibit high levels of variations related to insertions, deletions, microrepeats, and base substitutions. The phylogenetic analysis of the 5S rDNA sequences clustered the species into two clades that are in agreement with cytogenetic and morphological data.     

Evolution of multicellular animals as deduced from 5S rRNA sequences: a possible early emergence of the Mesozoa.

The nucleotide sequences of 5S rRNA from a mesozoan Dicyema misakiense and three metazoan species, i.e., an acorn-worm Saccoglossus kowalevskii, a moss-animal Bugula neritina, and an octopus Octopus vulgaris have been determined. A phylogenic tree of multicellular animals has been constructed from 73 5S rRNA sequences available at present including those from the above four sequences. The tree suggests that the mesozoan is the most ancient multicellular animal identified so far, its emergence time being almost the same as that of flagellated or ciliated protozoans. The branching points of planarians and nematodes are a little later than that of the mesozoan but are clearly earlier than other metazoan groups including sponges and jellyfishes. Many metazoan groups seem to have diverged within a relatively short period.     

Elongation factor 1-alpha sequences do not support an early divergence of the Acoela

The phylogenetic position of the Acoela is a key problem in the understanding of metazoan evolution. Recent studies based on 18S ribosomal DNA (rDNA) sequences have placed the Acoela in an extremely basal position as the sister group to all other extant triploblastic animals, suggesting that the phylum Platyhelminthes is polyphyletic. In order to test the results obtained with 18S rDNA, we sequenced elongation factor 1-alpha (EF1a) for the acoel Convoluta roscoffensis and five species of Turbellaria (two polyclads, Leptoplana tremellaris, and Prostheceraeus vittatus, and three triclads, Crenobia alpina, Schmidtea polychroa, and Girardia tigrina). Phylogenetic analyses of EF1a sequences show that the acoel sequences branch within the Platyhelminthes, in opposition to the 18S rDNA data. Moreover, comparison of the central variable region of EF1a shows similar sequence signatures between C. roscoffensis and the three triclad species. Although EF1a sequences fail to prove the monophyly of the phylum Platyhelminthes, they do not confirm the early divergence of the Acoela.     

Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata

The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.     

Molecular Organization of 5S rDNAs in Rajidae (Chondrichthyes): Structural Features and Evolution of Piscine 5S rRNA Genes and Nontranscribed Intergenic Spacers

The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]